Quinoline inhibitors of hyaki and hyak3 kinases

ABSTRACT

This invention relates to novel quinoline inhibitors of hYAK1 and hYAK3 kinases and pharmaceutically acceptable salts, hydrates or solvates thereof, pharmaceutical compositions thereof, and methods of treatment of diseases in which an excessive amount of either such kinase is a factor.

This This application is filed pursuant to 35 U.S.C. § 371 as a UnitedStates National Phase Application of International Application No.PCT/US02/10657 filed Apr. 4, 2002, which claims priority from U.S.60/282,229 filed Apr. 6, 2001.

FIELD OF THE INVENTION

This invention relates to novel quinoline inhibitors of hYAK kinases.Such compounds are particularly useful for treating disease states inwhich hYAK1 and/or hYAK3 kinases are implicated, especially diseases ofthe hematopoietic systems, including anemias due to renal insufficiencyor to chronic disease, such as autoimmunity or cancer and drug-inducedanemias, polycythemia, myelodysplastic syndrome, aplastic anemia andmyelosuppression; cytopenia; neurodegeneration; and also for controllingmale fertility, especially for the purpose of achieving contraception.

BACKGROUND OF THE INVENTION

The YAK family of serine/threonine protein kinases represent a novelfamily of dual specificity protein kinases with unique structural,enzymatic, and probably functional features was identified (Becker andJoost (1999) Prog. Nucl. Acid Res. 62, 1–17). Four members of thissubfamily have been identified by large scale screening of human cDNAlibraries using yeast YAK1 sequence, and have been termed h (human)Yak1,2, 3, and 4. See U.S. Pat. No. 5,972,606 (hYAK1), U.S. Pat. No.6,001,623 (hYAK2), and U.S. Pat. No. 5,965,420 (hYAK3). In the yeast S.cerevisiae YAK1 functions as a negative regulator of cell growth(Garrett, S., Menold, M. M., and Broach, J. (1991) Mol. Cell. Biol. 11,4045–4051). Deletion of the three PKA genes (tpk1, tpk2, and tpk3) inyeast causes cell cycle arrest at G₁ while this growth defect isalleviated by removal of the YAK1 gene (Garrett, S., and Broach, J.(1989) Gene Dev. 3, 1336–1348). Recent data indicates that yYAK1expression is controlled by two transcription factors MSN2/4 which arenegatively regulated by PKA, thus yYAK1 acts downstream of PKA (Smith,A., Ward, M. P. and Garrett, S. (1998) EMBO J. 17, 3556–3564). While themeans by which yYAK1 inhibits cell growth is still not known,overexpression of yYAK1 suppresses cell cycle arrest in late mitoticmutants activity (cdc15, cdc5, dbf2, and tem1) defective inanaphase-promoting complex (APC) (Jaspersen, S. L. Charles, J. F.,Tinker-Kulberg, R. L., and Morgan, D. O. (1998) Mol. Biol. of the Cell.9, 2803–2817). Recent work in Dictyostelium has uncovered a yYAK1homolog which is required for the transition from growth to developmentgiving support to the involvement of this family of kinases in cellgrowth (Souza, G. M., Lu, S. and Kuspa, A. (1998) Development 125,2291–2302).

Human multi-tissue northern blot analysis indicated that hYAK1 isexpressed as a ˜10 kb, 7.0 kb and 2.6 kb transcript. The multipletranscripts are not due to cross-hybridization with other YAK familymembers since the 3′UTR was used as a probe and the closest knownhomolog to hYAK1, hYAK3, shares only 62% identity with hYAK1 at thenucleotide level. In addition, alternatively spliced forms wereidentified within the 3′ UTR indicating that the multiple transcriptsare due to alternative splicing within the untranslated regions. Themost abundant transcripts were found in skeletal muscle and heartfollowed by pancreas, placenta, brain and lung. Multiple transcripts ofthe same apparent size were also seen in various osteoblastoid (HOS,MG63, Hob), stromal (TF274) and chondrocyte (C20A4) cell linesconfirming that hYAK1 is expressed in these tissues. In situhybridization studies were done using ³⁵S-labeled riboprobes oncryosections of human bone and giant cell tumor. Autoradiographicdevelopment times were extended (3 weeks) to compensate for thegenerally low level of mRNA expression of hYAK1 kinase observed in theinitial studies. In human fetal bone and osteophyte, various osteoblastpopulations were strongly (3+) positive for the expression of hYAK1kinase mRNA. Many other cell types including bone marrow andchondrocytes had varying levels of expression (1–2+). In giant celltumor, the diverse population of cell types including stromal,osteoblast precursors and osteoclasts were all positive (2+) for hYAK1kinase expression.

Several lines of evidence from our research findings strongly suggestthat hYAK1, like YAK1 in yeast functions as a negative regulator of cellcycle progression. Overexpression of wild type hYAK1 in cells causes adelay in exit from G2/M phase. Conversely, hYAK1 kinase inhibitorsselectively cause an accumulation of S phase cells. This in turn causeschanges in the expression of bone specific markers and products fromchondrocytes. Specifically, YAK1 inhibitors are expected to increasebone formation and/or to be chrondroprotective.

Northern analysis was carried out to determine the distribution of hYAK3mRNA in human tissues. Membranes containing mRNA from multiple humantissues (Clontech #7760-1, #7759-1, and #7768-1) were hybridized to anhYAK3 probe and washed under high stringency conditions as directed.Hybridized mRNA was visualized by exposing the membranes to X-ray film.One major transcript at ˜2.5 kb was present in testis, and transcriptsof 2.5, 8 and 10 kb were present in bone and fetal liver. Thetranscripts were not visible in any other tissues; however, dot blotanalysis using a Human Master blot (Clontech #7770-1) indicated thathYAK3 is expressed in other tissues including skeletal muscle.

Investigations with primary cells and hematopoietic cell lines from bothhuman and mouse indicate that cells of the erythroid lineage maypredominantly account for the elevated hYAK3 expression. These datasuggest that hYAK3 may have lineage-specific function. In cell lines,hYAK3 is present at higher levels in cells with an erythroid phenotypethan other hematopoietic lineages, including myeloid, monocytic andlymphoid cell lines. This profile is completely distinct from hYAK1which has been observed only at low constitutive levels in hematopoieticcells and tissues. EPO-treatment of human bone marrow in vitro leads toinduction and sustained expression of hYAK3 message and hYAK3 protein.Splenocytes from mice made anemic by phenylhydrazine treatment becomeenriched in erythroid progenitors and exhibit increased expression ofhYAK3. Increases in both message and protein accompany induction oferythroid differentiation in UT7-EPO cells.

In yeast, yYAK is a negative regulator of growth via the cell cycle.Consequently, we would anticipate that hYAK3 participates in cell cyclecontrol, and/or commitment to differentiation. We predict that anantagonist of hYAK3 would have a positive effect on cell growth. Ourdata indicates that it also may be involved in terminal differentiationand growth arrest in hematopoietic cells, especially in the erythroidlineage. Therefore compounds which antagonize YAK3 function or activitymay be therapeutically useful in treating conditions of hematopoieticcellular deficiency, such as anemias, including anemias due to renalinsufficiency or to chronic disease, such as autoimmunity or cancer,neutropenia, cytopenia, drug-induced anemias, polycythemia, cancer andmyelosuppression.

It now has been discovered that a certain novel quinoline inhibitors ofhYAK1 and/or hYAK3 kinases are useful for treating diseases of theerythroid and hematopoietic systems, including anemias due to renalinsufficiency or to chronic disease, such as autoimmunity or cancer anddrug-induced anemias, polycythemia, myelodysplastic syndrome, aplasticanemia and myelosuppression; cytopenia; neurodegeneration; and are alsouseful for controlling male fertility, especially for the purpose ofachieving contraception.

SUMMARY OF THE INVENTION

An object of the present invention is to provide novel quinolineinhibitors of hYAK1 and/or hYAK3 kinases. The compounds of the presentinvention are useful for treating diseases which may be therapeuticallymodified by altering the activity of such kinases.

Accordingly, in the first aspect, this invention provides a compound,according to Formula I.

In another aspect, this invention provides a pharmaceutical compositioncomprising a compound according to Formula I and a pharmaceuticallyacceptable carrier.

In yet another aspect, this invention provides a method of treatingdiseases in which the disease pathology may be therapeutically modifiedby inhibiting hYAK1 and/or hYAK3 kinases with compounds of Formula II,which include the compounds of Formula I. In particular, the methodincludes treating diseases by inhibiting the activity of such kinases.

In still another aspect, the compounds of this invention are especiallyuseful for treating diseases of the erythroid and hematopoietic systems,including anemias due to renal insufficiency or to chronic disease, suchas autoimmunity or cancer and drug-induced anemias, polycythemia,myelodysplastic syndrome, aplastic anemia and myelosuppression;cytopenia; and are also useful for controlling male fertility,especially for the purpose of contraception.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the activity of hYAK1, hYAK3, and yeast YAK1 against MBPand the Ser164 peptide. 5 ng purified hYAK1 and 100 ng purified hYAK3were used per assay. Anti-HA mAb immune complex kinase assay wasperformed on 100 ug protein from crude extracts of yeast cellsexpressing either FL or DN yeast YAK1. Concentration of ATP was 100 uM,Ser164 was used at 0.5 mM, and MBP was at 10 ug/reaction (18.5 uM).

FIG. 2 shows double reciprocal plots (1/V vs. 1/[substrate]) with S164peptide as the phosphate acceptor.

FIG. 3 shows 2-Chloro-7-methyl-quinoline-3-carboxylic acid enhancesCFU-E formation in the presence of erythropoietin. The number of CFU-Ecolonies recovered from human bone marrow cultures grown in the presenceof 2 U/ml erythropoietin and 0, 1 or 10 uM2-chloro-7-methyl-quinoline-3-carboxylic acid was measured.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a compound of Formula (I):

wherein:

-   -   R₁ is selected from the group consisting of: —NH—C₁₋₆alkyl,        —NH—C₃₋₇ cycloalkyl, —NH—C₃₋₇ cycloheteroalkyl, —NH-aryl,        —NH-Het, —O—C₁₋₆alkyl, —O—C₃₋₇ cycloalkyl, —O—C₃₋₇        cycloheteroalkyl, —O-aryl, —O-Het, —S—C₁₋₆ alkyl, —S—C₃₋₇        cycloalkyl, —S—C₃₋₇ cycloheteroalkyl, —S-aryl, —S-Het, —C₃₋₇        cycloalkyl and —C₃₋₇ cycloheteroalkyl;    -   R₂ is selected from the group consisting of: —CO₂H, —CONH₂,        —CHNOH, —CO₂R′, ——CH₂OH, —CHO, —CONHR″, —CONHCOR″, and        —CONHSO₂R″;    -   R₃ is selected from the group consisting of: —H, —OH,        —C₁₋₆alkyl, —C₃₋₇ cycloalkyl, aryl, Het, —O—C₁₋₆alkyl, —O—C₃₋₇        cycloalkyl, —O-aryl, —O-Het, —S—C₁₋₆alkyl, —S—C₃₋₇ cycloalkyl,        —S-aryl, —S-Het, —NH—C₁₋₆alkyl, —NH—C₃₋₇ cycloalkyl, —NH-aryl,        —NH-Het and halogen;    -   R₄ is selected from the group consisting of: —H, —C₁₋₆alkyl,        —C₃₋₇ cycloalkyl, aryl, Het, —O—C₁₋₆alkyl, —O—C₃₋₇ cycloalkyl,        —O-aryl, —O-Het, —S—C₁₋₆alkyl, —S—C₃₋₇ cycloalkyl,        —S-aryl,—S-Het, —NH—C₁₋₆alkyl, —NH—C₃₋₇ cycloalkyl, —NH-aryl,        —NH-Het and halogen;    -   R₃ and R₄ can form a 5 to 7 membered ring comprising 0–3        heteroatoms independently selected from the group consisting of:        O, N, and S;    -   R₅ is selected from the group —H and halogen;    -   R′ is selected from the group consisting of: —C₁₋₆alkyl,        —C₃₋₇cycloalkyl, and —C₃₋₇cycloheteroalkyl; and    -   R″ is selected from the group consisting of: —C₁₋₆alkyl,        —C₃₋₇cycloalkyl, —C₃₋₇cycloheteroalkyl, aryl, and Het;

or a pharmaceutically acceptable salt, hydrate or solvate thereof.

Preferred are compounds of Formula I wherein:

-   -   R₁ is preferably selected from the group consisting of:        —NH—C₁₋₆alkyl, —NH-aryl, —NH-Het, —O-aryl, —O-Het, —S-aryl,        —S-Het, and —C₃₋₇ cycloheteroalkyl,;    -   R₂ is preferably selected from the group consisting of: —CO₂H,        —CONH₂, and —CO₂R′;    -   R₃ is preferably selected from the group consisting of: —H,        —C₁₋₆alkyl, —O—C₁₋₆alkyl, —S—C₁₋₆alkyl, and halogen; and    -   R₄ is preferably selected from the group consisting of: —H,        —O—C₁₋₆alkyl, —S—C₁₋₆alkyl, and halogen.

When R₂ is —CO₂R′, R′ is preferably selected from the group consistingof: —C₁₋₆alkyl, and —C₃₋₇cycloalkyl.

More preferred are compounds of Formula I wherein:

-   -   R₁ is more preferably selected from the group consisting of:        —NH—C₁₋₆alkyl, —NH—aryl, —NH-Het, and —C₃₋₇ cycloheteroalkyl,;    -   R₂ is more preferably selected from the group consisting of:        —CO₂H and —CONH₂;    -   R₃ is more preferably selected from the group consisting of: —H,        —C₁₋₆alkyl, —O—C₁₋₆alkyl, —S—C₁₋₆alkyl, and halogen; and    -   R₄ is more preferably selected from the group consisting of: —H        and halogen, even more preferably R₄ is H; and    -   R₅ is more preferably —H.    -   Even more preferred are compounds of Formula I wherein, in R₁:        -   —NH-aryl is most preferably selected from the group            consisting of: methylphenylamino, especially            3-methylphenylamino (also known as m-tolylamino);            ethylphenylamino, especially 3-ethylphenylamino,            4-ethylphenylamino; cyclohexylphenylamino, especially            4-cyclohexylphenylamino; dimethylphenylamino, especially            3,4-dimethylphenylamino; chlorophenylamino, especially            2-chlorophenylamino, 3-chlorophenylamino,            4-chlorophenylamino; fluorophenylamino, especially            2-fluorophenylamino, 4-fluorophenylamino; iodophenylamino,            especially 4-iodophenylamino; chlorobenzylamino, especially            4-chlorobenzylamino; morpholinophenylamino,especially            4-morpholin-4-yl-phenylamino; cyanophenylamino, especially            3-cyanophenylamino, 4-cyanophenylamino; ethoxyphenylamino,            especially 4-ethoxyphenylamino; dimethoxyphenylamino,            especially 3,4-dimethoxyphenylamino, phenoxyphenylamino,            especially 4-phenoxyphenylamino; and            fluoro-ethoxyphenylamino, especially            2-fluoro-3-ethoxyphenylamino;        -   —NH-Het is most preferably selected from the group            consisting of: quinolinylamino, especially            quinolin-3-ylamino, quinolin-5-ylamino, quinolin-8-ylamino;            pyridinylamino, especially pyridin-3-ylamino; and            methoxy-pyridinylamino, especially            6-methoxy-pyridin-3-ylamino;        -   —C₃₋₇ cycloheteroalkyl is most preferably piperidino,            especially N-piperidino; and        -   —NH—C₁₋₆alkyl is preferably propylamino, especially            2-propylamino; and    -   in R₃:        -   —C₁₋₆alkyl is most preferably selected from the group            consisting of: methyl and ethyl;        -   —O—C₁₋₆alkyl is most preferably methoxy;        -   —S—C₁₋₆alkyl is most preferably methylsulfanyl; and        -   halogen is most preferably chloro.

Especially preferred compounds of the present invention are:

-   2-(3-Chloro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   7-Chloro-2-(3-chloro-phenylamino)-quinoline-3-carboxylic acid;    2-(3-Chloro-phenylamino)-7-methylsulfanyl-quinoline-3-carboxylic    acid;-   2-(4-Chloro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(3-Chloro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid amide-   2-(4-Chloro-benzylamino)-7-methoxy-quinoline-3-carboxylic acid;-   7-Methoxy-2-(4-phenoxy-phenylamino)-quinoline-3-carboxylic acid-   7-Methoxy-2-(4-morpholin-4-yl-phenylamino)-quinoline-3-carboxylic    acid;-   2-(3-Chloro-phenylamino)-quinoline-3-carboxylic acid;-   2-(3-Chloro-phenylamino)-7-methyl-quinoline-3-carboxylic acid;-   2-(3-Chloro-phenylamino)-6-methoxy-quinoline-3-carboxylic acid;-   2-(3-Chloro-phenylamino)-7-ethyl-quinoline-3-carboxylic acid;-   2-(3-Cyano-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   7-Methoxy-2-m-tolylamino-quinoline-3-carboxylic acid;-   2-(4-Ethoxy-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(4-Cyclohexyl-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(4-Fluoro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(2-Chloro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(4-Ethyl-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(3-Ethyl-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(4-Cyano-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   7-Methoxy-2-(quinolin-3-ylamino)-quinoline-3-carboxylic acid;-   2-(4-Iodo-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   7-Methoxy-2-(pyridin-3-ylamino)-quinoline-3-carboxylic acid;-   7-Methoxy-2-(6-methoxy-pyridin-3-ylamino)-quinoline-3-carboxylic    acid;-   7-Methoxy-2-(quinolin-8-ylamino)-quinoline-3-carboxylic acid;-   7-Methoxy-2-(quinolin-5-ylamino)-quinoline-3-carboxylic acid;-   2-(3,4-Dimethoxy-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(3,4-Dimethyl-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(2-Fluoro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;,-   2-(4-Ethoxy-2-fluoro-phenylamino)-7-methoxy-quinoline-3-carboxylic    acid;-   7-Methoxy-2-piperidin-1-yl-quinoline-3-carboxylic acid;    and7-Methoxy-2-propylamino-quinoline-3-carboxylic acid.

The compounds in the paragraph above may also be named as follows, inthe same order as above:

-   2-(3-chloroanilino)-3-carboxy-7-methoxy-quinoline;-   2-(3-chloroanilino)-3-carboxy-7-chloro-quinoline;    2-(3-chloroanilino)-3-carboxy-7-methylthio-quinoline;-   2-(4-chloroanilino)-3-carboxy-7-methoxy-quinoline;-   2-(3-chloroanilino)-3-carboxamido-7-methoxy-quinoline;-   2-(4-chlorobenzylamino)-3-carboxy-7-methoxy-quinoline;-   2-(4-phenoxyanilino)-3-carboxy-7-methoxy-quinoline;-   2-(4-morpholinanilino)-3-carboxy-7-methoxy-quinoline;-   2-(3-chloroanilino)-3-carboxy-quinoline;-   2-(3-chloroanilino)-3-carboxy-7-methyl-quinoline;-   2-(3-chloroanilino)-3-carboxy-6-methoxy-quinoline;-   2-(3-chloroanilino)-3-carboxy-7-ethyl-quinoline;-   2-(3-cyanoanilino)-3-carboxy-7-methoxy-quinoline;-   2-(3-methylanilino)-3-carboxy-7-methoxy-quinoline;-   2-(4-ethoxyanilino)-3-carboxy-7-methoxy-quinoline;-   2-(4-cyclohexylanilino)-3-carboxy-7-methoxy-quinoline;-   2-(4-fluoroanilino)-3-carboxy-7-methoxy-quinoline;-   2-(2-chloroanilino)-3-carboxy-7-methoxy-quinoline;-   2-(4-ethylanilino)-3-carboxy-7-methoxy-quinoline;-   2-(3-ethylanilino)-3-carboxy-7-methoxy-quinoline;-   2-(4-cyanoanilino)-3-carboxy-7-methoxy-quinoline;-   2-(3-aminoquinolino)-3-carboxy-7-methoxy-quinoline;-   2-(4-iodoanilino)-3-carboxy-7-methoxy-quinoline;-   2-(3-aminopyridino)-3-carboxy-7-methoxy-quinoline;-   2-(5-amino-2-methoxypyridino)-3-carboxy-7-methoxy-quinoline;-   2-(8-aminoquinolino)-3-carboxy-7-methoxy-quinoline;-   2-(5-aminoquinolino)-3-carboxy-7-methoxy-quinoline;-   2-(3,4-dimethoxyanilino)-3-carboxy-7-methoxy-quinoline;-   2-(3,4-dimethylanilino)-3-carboxy-7-methoxy-quinoline;-   2-(2-fluoroanilino)-3-carboxy-7-methoxy-quinoline;-   2-(2-fluoro-3-ethoxyanilino)-3-carboxy-7-methoxy-quinoline;-   2-piperidino-3-carboxy-7-methoxy-quinoline; and    2-propylamino-3-carboxy-7-methoxy-quinoline.

More Especially Preferred Compounds of the Present Invention are:

-   2-(3-Chloro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   7-Chloro-2-(3-chloro-phenylamino)-quinoline-3-carboxylic acid;    2-(3-Chloro-phenylamino)-7-methylsulfanyl-quinoline-3-carboxylic    acid;-   2-(4-Chloro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(4-Chloro-benzylamino)-7-methoxy-quinoline-3-carboxylic acid;-   7-Methoxy-2-(4-phenoxy-phenylamino)-quinoline-3-carboxylic acid;-   2-(3-Cyano-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   7-Methoxy-2-m-tolylamino-quinoline-3-carboxylic acid;-   2-(4-Ethoxy-phenylamino)-7-methoxy-quinoline-3-carboxylic acid-   2-(4-Cyclohexyl-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(4-Fluoro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(2-Chloro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(4-Ethyl-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(3-Ethyl-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(4-Cyano-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   7-Methoxy-2-(quinolin-3-ylamino)-quinoline-3-carboxylic acid;-   2-(4-Iodo-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   7-Methoxy-2-(6-methoxy-pyridin-3-ylamino)-quinoline-3-carboxylic    acid;-   7-Methoxy-2-(quinolin-8-ylamino)-quinoline-3-carboxylic acid;-   7-Methoxy-2-(quinolin-5-ylamino)-quinoline-3-carboxylic acid;-   2-(3,4-Dimethoxy-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(2-Fluoro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid; and-   2-(4-Ethoxy-2-fluoro-phenylamino)-7-methoxy-quinoline-3-carboxylic    acid.

Most Especially Preferred Compounds of the Present Invention are:

-   2-(3-Chloro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(3-Chloro-phenylamino)-7-methylsulfanyl-quinoline-3-carboxylic    acid;-   2-(4-Chloro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(3-Cyano-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   7-Methoxy-2-m-tolylamino-quinoline-3-carboxylic acid;-   2-(4-Ethoxy-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(4Fluoro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(2-Chloro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(4-Ethyl-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(3-Ethyl-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(4Cyano-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(4-Iodo-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   7-Methoxy-2-(quinolin-8-ylamino)-quinoline-3-carboxylic acid;-   7-Methoxy-2-(quinolin-5-ylamino)-quinoline-3-carboxylic acid;-   2-(2-Fluoro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid; and-   2-(4-Ethoxy-2-fluoro-phenylamino)-7-methoxy-quinoline-3-carboxylic    acid.

The present invention includes all hydrates, solvates, complexes,polymorphs and prodrugs of the compound of Formula (I). Prodrugs are anycovalently bonded compounds which release the active parent drugaccording to Formula (I) in vivo. Prodrugs of the compound of thepresent invention include ketone derivatives, specifically ketals orhemiketals.

All forms of isomers resulting from the presence of a chiral center inthe inventive compound, including enantiomers and diastereomers, areintended to be covered herein. The inventive compound may be used as aracemic mixture, an enantiomerically enriched mixture, or the racemicmixture may be separated using well-known techniques and an individualenantiomer may be used alone.

In the event that the present compound may exist in tautomeric forms,such as keto-enol tautomers, each tautomeric form is contemplated asbeing included within this invention whether existing in equilibrium orpredominantly in one form.

The present invention also provides a method of treatment of diseasescaused by pathological levels of either one or both YAK1 and YAK3kinases, which method comprises administering to an animal, particularlya mammal, most particularly a human, in need thereof one or morecompounds of Formula II. In addition to the above-identified compoundsof Formula I, the compounds of Formula II comprise compounds wherein R₁of Formula I is additionally selected from the group consisting of:halogen, C₁₋₆alkyl, and aryl.

Thus, the compounds of Formula II used in the present method may beconveniently defined as follows:

wherein:

-   -   R₆ is selected from the group consisting of: —NH—C₁₋₆alkyl,        —NH—C₃₋₇ cycloalkyl, —NH—C₃₋₇ cycloheteroalkyl, —NH-aryl,        —NH-Het, —O—C₁₋₆alkyl, —O—C₃₋₇ cycloalkyl, —O—C₃₋₇        cycloheteroalkyl, —O-aryl, —O-Het, —S—C₁₋₆ alkyl, —S—C₃₋₇        cycloalkyl, —S—C₃₋₇ cycloheteroalkyl, —S-aryl, —S-Het,        C₁₋₆alkyl, aryl,—C₃₋₇ cycloalkyl, —C₃₋₇ cycloheteroalkyl, and        halogen;    -   R₇ is selected from the group consisting of: —CO₂H, —CONH₂,        —CHNOH, —CO₂R′, —CH₂OH, —CHO, —CONHR″, —CONHCOR″, and        —CONHSO₂R″;    -   R₈ is selected from the group consisting of: —H, —OH,        —C₁₋₆alkyl, —C₃₋₇ cycloalkyl, aryl, Het, —O—C₁₋₆alkyl, —O—C₃₋₇        cycloalkyl, —O-aryl, —O-Het, —S—C₁₋₆alkyl, —S—C₃₋₇ cycloalkyl,        —S-aryl, —S-Het, —NH—C₁₋₆alkyl, —NH—C₃₋₇ cycloalkyl, —NH-aryl,        —NH-Het and halogen;    -   R₉ is selected from the group consisting of: —H, —C₁₋₆alkyl,        —C₃₋₇ cycloalkyl, aryl, Het, —O—C₁₋₆alkyl, —O—C₃₋₇ cycloalkyl,        —O-aryl, —O-Het, —S—C₁₋₆alkyl, —S—C₃₋₇ cycloalkyl, —S-aryl,        —S-Het, —NH—C₁₋₆alkyl, —NH—C₃₋₇ cycloalkyl, —NH-aryl, —NH-Het        and halogen;    -   R₈ and R₉ can form a 5 to 7 membered ring comprising 0–3        heteroatoms independently selected from the group consisting of:        O, N, and S;    -   R₁₀ is selected from the group consisting of: H and halogen;    -   R′ is selected from the group consisting of: C₁₋₆alkyl,        C₃₋₇cycloalkyl,    -   and C₃₋₇cycloheteroalkyl; and    -   R″ is selected from the group consisting of: C₁₋₆alkyl,        C₃₋₇cycloalkyl, C₃₋₇cycloheteroalkyl, aryl, and Het;    -   or a pharmaceutically acceptable salt, hydrate or solvate        thereof.        Preferably used in the present methods are the following        compounds of formula II wherein:    -   R₆ is preferably selected from the group consisting of: :        —NH—C₁₋₆alkyl, —NH-aryl, —NH-Het, —O-aryl, —O-Het, —S-aryl,        —S-Het, —C₃₋₇ cycloheteroalkyl, and halogen;    -   R₇ is preferably selected from the group consisting of: —CO₂H,        —CONH₂, and —CO₂R′;    -   R₈ is preferably selected from the group consisting of: —H,        —C₁₋₆alkyl, —O—C₁₋₆alkyl, —S—C₁₋₆alkyl, and halogen; and    -   R₉ is preferably selected from the group consisting of: —H,        —O—C₁₋₆alkyl, —S—C₁₋₆alkyl, and halogen.

When R₇ is —CO₂R′, R′ is preferably selected from the group consistingof: —C₁₋₆alkyl, and —C₃₋₇cycloalkyl.

More preferably used in the present methods are compounds of formula IIwherein:

-   -   R₆ is more preferably selected from the group consisting of:        —NH—C₁₋₆alkyl, —NH-aryl, —NH-Het, —C₃₋₇ cycloheteroalkyl and        halogen;    -   R₇ is more preferably selected from the group consisting of:        —CO₂H and —CONH₂;    -   R₈ is more preferably selected from the group consisting of: —H,        —C₁₋₆alkyl, —O—C₁₋₆alkyl, —S—C₁₋₆alkyl, and halogen; and    -   R₉ is more preferably selected from the group consisting of: —H        and halogen, even more preferably R₉ is H; and    -   R₁₀ is more preferably —H.        When, in R₆, halogen is chlorine:    -   R₇ is selected from the group consisting of: —CO₂H, —CONH₂,        —CHNOH and —CO₂R′;    -   R₈ is selected from the group consisting of: —H, —OH,        —C₁₋₆alkyl, —O—C₁₋₆alkyl, —O—C₃₋₇ cycloalkyl, —S—C₁₋₆alkyl, and        halogen;    -   R₉ is selected from the group consisting of: —H, —C₁₋₆alkyl,        —O—C₁₋₆alkyl, most preferably wherein —C₁₋₆alkyl is —CH₃,        —S—C₁₋₆alkyl, most preferably wherein —C₁₋₆alkyl is —CH₃, and        halogen; and    -   R₁₀ is selected from the group consisting of: —H and halogen.        Even more preferred are compounds of Formula II used in the        present method wherein, in R₆:    -   NH-aryl is most preferably selected from the group consisting        of: methylphenylamino, especially 3-methylphenylamino (also        known as m-tolylamino); ethylphenylamino, especially        3-ethylphenylamino, 4-ethylphenylamino; cyclohexylphenylamino,        especially 4-cyclohexylphenylamino; dimethylphenylamino,        especially 3,4-dimethylphenylamino; chlorophenylamino,        especially 2-chlorophenylamino, 3-chlorophenylamino,        4-chlorophenylamino; fluorophenylamino, especially        2-fluorophenylamino, 4-fluorophenylamino; iodophenylamino,        especially 4-iodophenylamino; chlorobenzylamino, especially        4-chlorobenzylamino; morpholinophenylamino,especially        4-morpholin-4-yl-phenylamino; cyanophenylamino, especially        3-cyanophenylamino, 4-cyanophenylamino; ethoxyphenylamino,        especially 4-ethoxyphenylamino; dimethoxyphenylamino, especially        3,4-dimethoxyphenylamino, phenoxyphenylamino, especially        4-phenoxyphenylamino; and fluoro-ethoxyphenylamino, especially        2-fluoro-3-ethoxyphenylamino;    -   —NH-Het is most preferably selected from the group consisting        of: quinolinylamino, especially quinolin-3-ylamino,        quinolin-5-ylamino, quinolin-8-ylamino; pyridinylamino,        especially pyridin-3-ylamino; and methoxy-pyridinylamino,        especially 6-methoxy-pyridin-3-ylamino;    -   —C₃₋₇ cycloheteroalkyl is most preferably piperidino, especially        N-piperidino;    -   —NH—C₁₋₆alkyl is preferably propylamino, especially        2-propylamino; and    -   halogen is preferably chloro; and        in R₈:    -   —C₁₋₆alkyl is most preferably selected from the group consisting        of: methyl and ethyl;    -   —O—C₁₋₆alkyl is most preferably methoxy;    -   —S—C₁₋₆alkyl is most preferably methylsulfanyl; and    -   halogen is most preferably chloro.

Especially preferred compounds of the present invention for use in thepresent methods are:

-   2-(3-Chloro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-Chloro-7-methoxy-quinoline-3-carboxylic acid;    7-Chloro-2-(3-chloro-phenylamino)-quinoline-3-carboxylic acid;    2-(3-Chloro-phenylamino)-7-methylsulfanyl-quinoline-3-carboxylic    acid;-   2-(4-Chloro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(3-Chloro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid amide-   2-(4-Chloro-benzylamino)-7-methoxy-quinoline-3-carboxylic acid;-   7-Methoxy-2-(4phenoxy-phenylamino)-quinoline-3-carboxylic acid-   7-Methoxy-2-(4-morpholin-4-yl-phenylamino)-quinoline-3-carboxylic    acid;-   2-(3-Chloro-phenylamino)-quinoline-3-carboxylic acid;-   2-(3-Chloro-phenylamino)-7-methyl-quinoline-3-carboxylic    acid;2-Chloro-7-methyl-quinoline-3-carboxylic acid;-   2-(3-Chloro-phenylamino)-6-methoxy-quinoline-3-carboxylic acid;-   2-(3-Chloro-phenylamino)-7-ethyl-quinoline-3-carboxylic acid;-   2-(3-Cyano-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   7-Methoxy-2-m-tolylamino-quinoline-3-carboxylic acid;-   2-(4-Ethoxy-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(4-Cyclohexyl-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(4-Fluoro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(2-Chloro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(4-Ethyl-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(3-Ethyl-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(4-Cyano-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   7-Methoxy-2-(quinolin-3-ylamino)-quinoline-3-carboxylic acid;-   2-(4-Iodo-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   7-Methoxy-2-(pyridin-3-ylamino)-quinoline-3-carboxylic acid;-   7-Methoxy-2-(6-methoxy-pyridin-3-ylamino)-quinoline-3-carboxylic    acid;-   7-Methoxy-2-(quinolin-8-ylamino)-quinoline-3-carboxylic acid;-   7-Methoxy-2-(quinolin-5-ylamino):quinoline-3-carboxylic acid;-   2-(3,4-Dimethoxy-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(3,4-Dimethyl-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(2-Fluoro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(4-Ethoxy-2-fluoro-phenylamino)-7-methoxy-quinoline-3-carboxylic    acid;-   7-Methoxy-2-piperidin-1-yl-quinoline-3-carboxylic acid;    and7-Methoxy-2-propylamino-quinoline-3-carboxylic acid.

2-Chloro-7-methoxy-quinoline-3-carboxylic acid may also be named2-chloro-3-carboxy-7-methoxy-quinoline; and2-chloro-7-methyl-quinoline-3-carboxylic acid; may also be named2-chloro-3-carboxy-7-methyl-quinoline.

More especially preferred compounds of the present invention for use inthe present Methods are:

-   2-(3-Chloro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   7-Chloro-2-(3-chloro-phenylamino)-quinoline-3-carboxylic acid;    2-(3-Chloro-phenylamino)-7-methylsulfanyl-quinoline-3-carboxylic    acid;-   2-(4-Chloro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(4-Chloro-benzylamino)-7-methoxy-quinoline-3-carboxylic acid;-   7-Methoxy-2-(4-phenoxy-phenylamino)-quinoline-3-carboxylic acid;-   2-Chloro-7-methyl-quinoline-3-carboxylic acid;-   2-(3-Cyano-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   7-Methoxy-2-m-tolylamino-quinoline-3-carboxylic acid;-   2-(4-Ethoxy-phenylamino)-7-methoxy-quinoline-3-carboxylic acid-   2-(4-Cyclohexyl-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(4-Fluoro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(2-Chloro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(4-Ethyl-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(3-Ethyl-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(4-Cyano-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   7-Methoxy-2-(quinolin-3-ylamino)-quinoline-3-carboxylic acid;-   2-(4-Iodo-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   7-Methoxy-2-(6-methoxy-pyridin-3-ylamino)-quinoline-3-carboxylic    acid;-   7-Methoxy-2-(quinolin-8-ylamino)-quinoline-3-carboxylic acid;-   7-Methoxy-2-(quinolin-5-ylamino)-quinoline-3-carboxylic acid;-   2-(3,4-Dimethoxy-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(2-Fluoro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid; and-   2-(4-Ethoxy-2-fluoro-phenylamino)-7-methoxy-quinoline-3-carboxylic    acid.    Most especially preferred compounds of the present invention for use    in the present methods are:-   2-(3-Chloro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(3-Chloro-phenylamino)-7-methylsulfanyl-quinoline-3-carboxylic    acid;-   2-(4-Chloro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-Chloro-7-methyl-quinoline-3-carboxylic acid;-   2-(3-Cyano-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   7-Methoxy-2-m-tolylamino-quinoline-3-carboxylic acid;-   2-(4-Ethoxy-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(4-Fluoro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(2-Chloro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(4-Ethyl-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(3-Ethyl-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(4-Cyano-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   2-(4-Iodo-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;-   7-Methoxy-2-(quinolin-8-ylamino)-quinoline-3-carboxylic acid;-   7-Methoxy-2-(quinolin-5-ylamino)-quinoline-3-carboxylic acid;-   2-(2-Fluoro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid; and-   2-(4-Ethoxy-2-fluoro-phenylamino)-7-methoxy-quinoline-3-carboxylic    acid.

DEFINITIONS

“hYAK1 kinase” means human YAK 1 kinase.

“hYAK3 kinase” means human YAK3 kinase.

“C₁₋₆alkyl” as applied herein is meant to include substituted andunsubstituted methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl andt-butyl, pentyl, n-pentyl, isopentyl, neopentyl and hexyl and the simplealiphatic isomers thereof. Any C₁₋₆alkyl group may be optionallysubstituted independently by one to five halogens, SR′″, OR′″, orN(R′″)₂, where R′″ is C₁₋₆alkyl.

“C₃₋₇ cycloalkyl” as applied herein is meant to include substituted andunsubstituted cyclopropane, cyclobutane, cyclopentane, cyclohexane andcycloheptane.

“C₃₋₇ cycloheteroalkyl” as applied herein is meant to include 3-, 4-,5-, 6-, and 7-membered rings having at least one, but no more thanthree, ring heteroatom(s) selected from the group consisting of: N, O,and S. Examples include, but are not limited to, piperidine, piperazine,morpholine, thiomorpholine, tetrahydrofuran, tetrahydrothiophene,pyrrolidine, azepane, oxepane, and thiepane.

“Halogen” means F, Cl, Br, and I.

“Ar” or “aryl” means phenyl, benzyl or naphthyl, optionally substitutedby one or more of Ph, Het, Ph-C₀₋₆alkyl; Het-C₀₋₆alkyl; C₁₋₆alkoxy;Ph-C₀₋₆alkoxy; Het-C₀₋₆alkoxy; OH, (CH₂)₁₋₆NR¹¹R¹²; O(CH₂)₁₋₆NR¹¹R¹²;C₁₋₆alkyl, C₃₋₇cycloalkyl, OR″″, N(R″″)₂, SR″″, CF₃, NO₂, CN, CO₂R″″,CON(R″″), F, Cl, Br or I; where R¹¹ and R¹² are H, C₁₋₆alkyl,Ph-C₀₋₆alkyl, naphthyl-C₀₋₆alkyl or Het-C₀₋₆alkyl; and R″″ is H, phenyl,naphthyl, Het or C₁₋₆alkyl.

The term “—N—C₁₋₆alkyl” includes both mono- and di-C₁₋₆alkylsubstitutions on the N, including di-substitutions resulting in anN-containing cyclic ring, e.g.,

As used herein “Het” or “heterocyclic” represents a stable 5- to7-membered monocyclic, a stable 7- to 10-membered bicyclic, or a stable11- to 18-membered tricyclic heterocyclic ring which is either saturatedor unsaturated, and which consists of carbon atoms and from one to threeheteroatoms selected from the group consisting of N, O and S, andwherein the nitrogen and sulfur heteroatoms may optionally be oxidized,and the nitrogen heteroatom may optionally be quaternized, and includingany bicyclic group in which any of the above-defined heterocyclic ringsis fused to a benzene ring. The heterocyclic ring may be attached at anyheteroatom or carbon atom which results in the creation of a stablestructure, and may optionally be substituted with one or two moietiesselected from C₀₋₆Ar, C₁₋₆alkyl, OR¹³, N(R¹³)₂, SR¹³, CF₃, NO₂, CN,CO₂R¹³, CON(R¹³), F, Cl, Br and I, where R¹³ is —H, phenyl, naphthyl, orC₁₋₆alkyl. Examples of such heterocycles include, but are not limitedto, piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl,2-oxopyrrolodinyl, 2-oxoazepinyl, azepinyl, pyrrolyl, 4-piperidonyl,pyrrolidinyl, pyrazolyl, pyrazolidinyl, imidazolyl, pyridinyl,pyrazinyl, oxazolidinyl, oxazolinyl, oxazolyl, isoxazolyl, morpholinyl,thiazolidinyl, thiazolinyl, thiazolyl, quinuclidinyl, indolyl,quinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl, benzoxazolyl,furyl, pyranyl, tetrahydrofuryl, tetrahydropyranyl, thienyl,benzoxazolyl, thiamorpholinyl sulfoxide, thiamorpholinyl sulfone, andoxadiazoly, as well as triazolyl, thiadiazolyl, oxadiazolyl, isoxazolyl,isothiazolyl, imidazolyl, pyridazinyl, pyrimidinyl, triazinyl andtetrazinyl which are available by routine chemical synthesis and arestable. The term heteroatom as applied herein refers to oxygen, nitrogenand sulfur.

The term “R₃ and R₄ (as well as “R₈ and R₉”) can form a 5 to 7 memberedring comprising 0–3 heteroatoms independently selected from the groupconsisting of: O, N, and S″ includes, but is not limited to:methylenedioxy, imadazoyly, pyrrolyl, dihydropyrrolyl, thiophenyl,dihydrothiophenyl, furanyl, dihydrofuranyl or triazinyl.

Certain radical groups are abbreviated herein. Thus, t-Bu refers to thetertiary butyl radical, Ph refers to the phenyl radical.

Certain reagents are abbreviated herein. DMF refers to dimethylformamide, and DMSO refers to dimethyl sulfoxide.

METHOD OF PREPARATION

Methods for preparing compounds of the Formula I are shown in Schemes1–3

In general, the synthetic methods used herein are those detailed in J.C. S. Perkin 1, 1981, 5, 1520–30 and J. Het. Chem. 1991, 28(5), 1339–40for preparing substituted carboxy quinolines. Briefly, a substitutedaniline (I) is acylated with acetic anhydride in pyridine to give theresulting acetanilide (II). Treatment of the acetanilide (II) with POCl₃in DMF gives a 2-chloride-3-formyl quinoline (III). Oxidation with AgNO₃in basic ethanol gives the corresponding 2-chloride-3-carboxy quinoline(IV). The 2-chloro can be replaced with either aryl or alkyl amines inDMSO to give the resulting 2-substituted quinoline (V) (Scheme 1).

Alternatively, the novel method of Scheme 2 may be used, in which the2-chloride-3-carboxy quinoline (IV) is treated with an aryl amine in thepresence of excess of lithium hexamethyldisilazane in THF (−70 C to RT)to give the 2-substituted quinoline (V) (Scheme 2). For alkyl amines,excess of the lithium salt of the particular alkyl amine is used inplace of lithium hexamethyldisilazane. The novel method is furtherdisclosed in Example 13.

In the cases where R₂ is alkyl or aryl, the aniline I can be treatedwith POCl₃ and the keto ester VI to give VII which is subsequentlyconverted by hydrolysis to VII.

The starting materials used herein are commercially available or areprepared by routine methods well known to those of ordinary skill in theart and can be found in standard reference books, such as the Compendiumof Organic Synthetic Methods, Vol. I–VI (published byWiley-Interscience).

Acid addition salts of the compound of Formula I are prepared in astandard manner in a suitable solvent from the parent compound and anexcess of an acid, such as hydrochloric, hydrobromic, hydrofluoric,sulfuric, phosphoric, acetic, trifluoroacetic, maleic, succinic ormethanesulfonic acid.

UTILITY OF THE PRESENT INVENTION

This invention also provides a pharmaceutical composition whichcomprises at least one compound according to Formula I and apharmaceutically acceptable carrier, excipient or diluent. Thesepharmaceutical compositions are useful in the methods of treatment ofthis invention. Pharmaceutical compositions comprising at least onecompound of Formula II and a pharmaceutically acceptable carrier,excipient or diluent are also useful in the methods of treatment of thisinvention. Accordingly, at least one compound of Formula I or Formula IImay be used in the manufacture of a medicament. Pharmaceuticalcompositions of a compound of Formula I or Formula II prepared ashereinbefore described may be formulated as solutions or lyophilizedpowders for parenteral administration. Powders may be reconstituted byaddition of a suitable diluent or other pharmaceutically acceptablecarrier prior to use. The liquid formulation may be a buffered,isotonic, aqueous solution. Examples of suitable diluents are normalisotonic saline solution, standard 5% dextrose in water, or bufferedsodium or ammonium acetate solution. Such formulation is especiallysuitable for parenteral administration, but may also be used for oraladministration or contained in a metered dose inhaler or nebulizer forinsufflation. It may be desirable to add excipients such aspolyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia, polyethyleneglycol, mannitol, sodium chloride, or sodium citrate.

Alternately, this compound may be encapsulated, tableted, or prepared inan emulsion or syrup for oral administration. Pharmaceuticallyacceptable solid or liquid carriers may be added to enhance or stabilizethe composition, or to facilitate preparation of the composition. Solidcarriers include starch, lactose, calcium sulfate dihydrate, terra alba,magnesium stearate or stearic acid, talc, pectin, acacia, agar orgelatin. Liquid carriers include syrup, peanut oil, olive oil, salineand water. The carrier may also include a sustained release materialsuch as glyceryl monostearate or glyceryl distearate, alone or with awax. The amount of solid carrier varies but, preferably, will be betweenabout 20 mg to about 1 g per dosage unit. The pharmaceuticalpreparations are made following the conventional techniques of pharmacyinvolving milling, mixing, granulating, and compressing, when necessary,for tablet forms; or milling, mixing and filling for hard gelatincapsule forms. When a liquid carrier is used, the preparation will be inthe form of a syrup, elixir, emulsion or an aqueous or non-aqueoussuspension. Such a liquid formulation may be administered directly orfilled into a soft gelatin capsule.

For rectal administration, the compound of this invention may also becombined with excipients such as cocoa butter, glycerin, gelatin orpolyethylene glycols and molded into a suppository.

Compounds of Formula I and Formula II are useful as inhibitors of eitherone or both hYAK1 and hYAK3 kinases. The present invention also providesuseful compositions and formulations of said compounds, includingpharmaceutical compositions and formulations of said compounds.

The present compounds are useful for treating disease states in whicheither one or both hYAK1 and hYAK3 kinases are implicated, especiallydiseases of the hematopoietic system, including anemias due to renalinsufficiency or to chronic disease, such as autoimmunity or cancer anddrug-induced anemias, polycythemia, myelodysplastic syndrome, aplasticanemia and myelosuppression; cytopenia; and are also useful forcontrollinging male fertility, especially for the purpose of achievingcontraception.

The present invention also provides methods of treatment of diseasescaused by pathological levels of either one or both YAK1 and YAK3kinases, especially diseases of the hematopoietic system, includinganemias due to renal insufficiency or to chronic disease, such asautoimmunity or cancer and drug-induced anemias, polycythemia,myelodysplastic syndrome, aplastic anemia and myelosuppression;cytopenia; and also a method of controllinging male fertility,especially for the purpose of achieving contraception, which methodscomprise administering to an animal, particularly a mammal, mostparticularly a human, in need thereof one or more compounds of FormulaII.

The present method is especially useful in treating diseases of thehematopoietic system, particularly anemias. Such anemias include ananemia selected from the group comprising: aplastic anemia andmyelodysplastic syndrome. Such anemias also include those wherein theanemia is a consequence of a primary disease selected from the groupconsisting of: cancer, leukemia and lymphoma. Such anemias also includethose wherein the anemia is a consequence of a primary disease selectedfrom the group consisting of: renal disease, failure or damage. Suchanemias include those wherein the anemia is a consequence ofchemotherapy or radiation therapy, in particular wherein thechemotherapy is chemotherapy for cancer or AZT treatment for HIVinfection. Such anemias include those wherein the anemia is aconsequence of a bone marrow transplant or a stem cell transplant. Suchanemias also include anemia of newborn infants. Such anemias alsoinclude those which are a consequence of viral, fungal, microbial orparasitic infection.

The present invention provides a method of enhancement of normal redblood cell numbers. Such enhancement is desireable for a variety ofpurposes, especially medical purposes such as preparation of a patientfor transfusion and preparation of a patient for surgery.

The present invention also provides a method of lowering normal levelsof either one or both hYAK1 and hYAK3 to achieve a desired clinicaleffect, especially controlling male fertility to achieve contraception.

In accordance with this invention, an effective amount of a compound ofFormula II is administered to inhibit the hYAK1 and/or hYAK3 kinaseimplicated in a particular condition or disease. Of course, this dosageamount will further be modified according to the type of administrationof the compound. For example, for acute therapy, parenteraladministration of the compound of Formula II is preferred. Anintravenous infusion of the compound in 5% dextrose in water or normalsaline, or a similar formulation with suitable excipients, is mosteffective, although an intramuscular bolus injection is also useful.Typically, the parenteral dose will be about 0.01 to about 100 mg/kgpreferably between 0.1 and 10 mg/kg, in a manner to maintain theconcentration of drug in the plasma at a concentration effective toinhibit hYAK1 and/or hYAK3. The compound is administered one to fourtimes daily at a level to achieve a total daily dose of about 0.4 toabout 400 mg/kg/day. The precise amount of an inventive compound whichis therapeutically effective, and the route by which such compound isbest administered, is readily determined by one of ordinary skill in theart by comparing the blood level of the agent to the concentrationrequired to have a therapeutic effect.

Prodrugs of the compounds of the present invention may be prepared byany suitable method. Where the prodrug moiety is a ketone functionality,specifically ketals and/or hemiacetals, the conversion may be effectedin accordance with conventional methods.

The compounds of this invention may also be administered orally to thepatient, in a manner such that the concentration of drug is sufficientto inhibit bone resorption or to achieve any other therapeuticindication as disclosed herein. Typically, a pharmaceutical compositioncontaining the compound is administered at an oral dose of between about0.1 to about 100 mg/kg in a manner consistent with the condition of thepatient. Preferably the oral dose would be about 0.5 to about 20 mg/kg.No unacceptable toxicological effects are expected when compounds of thepresent invention are administered in accordance with the presentinvention.

BIOLOGICAL ASSAYS

The compounds of this invention may be tested in one of severalbiological assays to determine the concentration of compound which isrequired to have a given pharmacological effect.

Kinase Assays Using Ser164 as the Phosphoacceptor

The source of Ser164 peptide The Ser164 (LGGRDSRSGSPMARR-OH) peptide waspurchased from California Peptide Research Inc. (Napa, Calif.), and itspurity was determined by HPLC. The peptide contained 15 amino acids, andits calculated molecular mass was 1601.82 dalton. Solid sample wasdissolved at 5 mM in ice-cold kinase assay buffer (see later),aliquoted, and stored at −20° C. until use.

The source of enzyme:

1) hYAK1: DET1/DET2-tagged full length hYAK1 was expressed in Drosophilasf9 cells and purified to >95% purity using Ni column chromatography.The purified protein migrated on SDS gels as a single band an apparentmolecular mass of 62 kDa. Samples were stored at −80° C. until use.

2) hYAK3: Glutathione-S-Transferase (GSI)/Factor Xa-tagged hYAK3B wasexpressed in baculovirus cells and purified to about 50% purity usingGlutathione Sepharose 4B column chromatography, followed by Ni-NTAcolumn chromatography. Samples were stored at −80° C. until use.

3) Yeast YAK1: Full length and an amino-terminally truncated (aminoacids 148–807, termed ΔN) hemagglutinin (HA)-tagged yeast YAK1 was eachexpressed in a strain of S. cerevisiae lacking the endogenous YAK1 geneand all three PKA genes. Cultures for experiments were grown in liquidSc-His to an OD₆₀₀ of at least 1.0, washed with Sc-His g/r, resuspendedin Sc-His g/r to twice the original volume and grown for 16–24 h at RT.Cells were washed once with H₂O and the pellets stored at −80° C. untiluse. To prepare lysates, cell pellets were thawed and resuspended at 1ml/100 ml of original culture in lysis buffer (LB) containing 50 mM TrispH 7.5, 150 mM NaCl, 10 μg/ml each aprotinin, leupeptin and TLCK, 0.1 mMPMSF, 50 mM NaF, 1 mM Na vanadate, 10 mM β-glycerophosphate. Followingthe addition of 0.5 ml sterile acid-washed glass beads, cells weredisrupted via ten, 30 sec intervals of vortexing. NP40 was added to a 2%final concentration followed by rocking at 4° C. for 30–50 min. Lysateswere clarified by high speed centrifugation, and the supernatants werestored at −80° C. until use. Each form of yeast YAK1 wasimmunoprecipitated from the detergent extracts using anti-HA mAb.

Immune Complex Protein Kinase Assay for Yeast YAK1: Yeast cellularextracts were immunoprecipitated by rocking overnight at 4° C. with 4 μgof the anti-HA tag antibody and 100 μl of 20% suspension of protein Aagarose (GIBCO-BRL) in LB that contained 1% NP-40. Samples were thenwashed twice with LB and once with basic kinase assay buffer (25 mMHepes, pH 7.5; 1 mM DTT; 10 mM β-glycerol phosphate; 0.2 mM NaV). Washedimmune complexes were suspended in 20 μl of basic kinase assay bufferthat contained 0.1 mM ATP, 3 μCi of [γ-³²P]ATP, 10 MM MgCl₂, plus eitherbovine MBP or the Ser164 peptide. After incubation for 15 min at 30° C.,the reactions were stopped by adding 20 μl of 0.15 M phosphoric acid.Phosphorylated substrates were isolated by spotting 20 μl of each sampleon phosphocellulose (p81) filters. Filters were washed 3 times with 75mM phosphoric acid followed by 3 times with H₂O, and counted for ³²Pincorporation using β-scintillation counter.

Kinase assay of purified hYAK1 and hYAK3: Assay was performed in 96 wellMinisorp plates (Costar, Catalog No. 3356). Reactions (in 30 ul volume)mix contained in final concentrations 25 mM Hepes buffer, pH 7.5; 0.2 mMsodium vanadate; 10 MM MgCl₂; 1 mM DTT; 10 mM β-glycerol phosphate; 0.1%BSA; 0.1 mM ATP, 2.5 μCi of [γ-³²P]ATP; purified hYAK1 (1–5 ng/assay),or purified hYAK3 (50–100 ng/assay); and either bovine MBP or the Ser164peptide used at the concentrations indicated below and the legends tofigures. Reactions were incubated for 20 min at 37° C., and were stoppedby adding 10 μl of 0.3 M phosphoric acid. Phosphorylated substrates wereisolated by spotting 20 μl of p81 filters, and processed as detailedearlier.

This same assay can be performed on a FlashPlate format in which theplate is coated with MBP or with the S164 peptide by incubationovernight at 4° C. in 100 ul of either substrate dissolved in SodiumCarbonate buffer, pH 8.8. When coating with MBP, a solution of 100 ug/mlMBP was used to coat wells with 100 ul (10 ug) MBP per well. Whencoating with Ser164, a solution of 0.4 mg/ml (0.25 mM) was used to coatwells with 100 ul (40 ug) Ser164 per well. An example of a FlashPlateassay protocol and typical results are given below:

FlashPlate Protocol

-   1. Coat Maxisorp plates (Nunk, Immunoplate, Maxisorp™) with MBP or    Ser164 as above.-   2. Wash plates once with kinase assay buffer (KB): 25 mM Hepes, pH    7.5; 0.2 mM NaV; 10 mM.-glycerol phosphate; 1 mM Na pyrophosphate-   3. Add enzyme (Ni-hYAK1, diluted in KB), DMSO or inhibitors (in KB)    and keep on ice 30 min-   4. Add KB containing Mg/AJP to a [final] of 0.1 mM [.-33P]ATP and 10    mM MgCl₂-   5. Incubate with shaking, 1–2 hrs, RT-   6. Aspirate and wash 6×0.5 ml KB-   7. Read 33P incorporation in FP reader-   8. Blank=No enzyme added-   9. Reaction volume: 25, 50 or 100 ul-   10. 0.5 or 1.0 uCi 33P/0.1 mM ATP-   11. MBP-FP better than basic FP (in house coating)-   12. 37° C. incubation was not better (several time points)-   13. Other incubation times at RT were not better    Results:

Each kinase phosphorylated the Ser164 peptide with much higher specificactivity than MBP (FIG. 1). Steady state kinetic constants of hYak1reaction were generated by varying both substrates simultaneously andfitting enzyme velocity as a function of each substrate concentration.Double reciprocal plots (1/V vs. 1/[Substrate]) with S164 peptide as thephosphate acceptor are shown in FIG. 2. GraphFit analysis of the resultsgenerated the following steady state kinetic constants:K _(m) [ATP]=42±7 uM.K _(m) [S164]=160±14 uM.V _(max)=51±6 umol/mg.k _(cat)=160±19 min⁻¹.Typical results of FlashPlate are shown below

FlashPlate Typical Results

Signal to noise ratio: >7 fold Blanks: 30–80 cpm [Ni-hYAK1]: As low as20 ng/reaction (5 nM) for 100 ul reactions As low as 8 ng/reaction (5nM) for 25 ul reactions 33P: As low as 0.5 uCi Kinase inhibitors:Potency comparable to tube assay: SKF-108752 IC50: 0.1 ug hYAK1: 0.19 uM0.3 ug hYAK1: 0.13 uM; 0.16 uM K252a IC50 (0.3 ug hYAK1): 0.552 uM;0.427 uM Specific Activity: At 20 ng enzyme, MBP gave 58 ± 3 (n = 6),and Ser164 gave 484 ± 63 nmol/mg protein (n = 6), DMSO: No effect up to3% Variability: <10% (between wells and from plate to plate).

The IC₅₀ of the present compounds, as measured in the assays describedabove, respecting hYAK1 is about 0.01 to about 10 uM, and about 0.03 toabout 10 uM respecting hYAK3.

The skilled artisan would consider any compound exhibiting an IC₅₀ valueof less than 1 uM to be a potential lead compound for further research,and an inhibitor exhibiting an IC₅₀ of less than 0.05 uM to be a drugdevelopment drug candidate assuming an acceptable pathology/toxicologyprofile and in vivo activity.

Human Colony Forming Unit-Erythroid (CFU-E) Assay

Light density cells from human bone marrow centrifuged over Histopaque1077 were washed and resuspended at 2.5×10⁶ cells/ml in X-vivo medium. Afinal concentration of: cells (2.5×105/ml), fetal calf serum (25%),bovine serum albumin (1%) and methylcellulose (0.8%) in X-Vivo mediumwere added in a volume of 0.4 ml per well of a 24-well TC dish (Nunc).The compound of the present invention was diluted in X-vivo medium andadded at final concentrations of 1 and 10 uM to the wells. All wellscontained 2 U/ml erythropoietin (EPO). The cultures were incubated at37°, 5% CO₂, 5% O₂ for seven days. Colonies were identified bymicroscopic examination as a group of greater than eight red,hemoglobinized cells.

Results:

The addition of 2-chloro-7-methyl-quinoline-3-carboxylic acid to humanbone marrow cultures enhanced the recovery of erthyroid colonies in theCFU-E assay (FIG. 3). In the presence of 2 U/ml erythropoietin, 10 uM of2-chloro-7-methyl-quinoline-3-carboxylic acid enhances CFU-E recovery by50%.

Compounds of the present invention which enhance CFU-E recovery in thisassay may be useful for treatment of diseases of the hematopoieticsystem, as disclosed herein above.

EXAMPLES

In the following synthetic examples, unless otherwise indicated, all ofthe starting materials were obtained from commercial sources. Withoutfurther elaboration, it is believed that one skilled in the art can,using the preceding description, utilize the present invention to itsfullest extent. These Examples are given to illustrate the invention,not to limit its scope. Reference is made to the claims for what isreserved to the inventors hereunder.

Example 1 Preparation of2-(3-Chloro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid

(a) 2-Chloro-3-formyl-7-methoxy-quinoline

The title compound was prepared using the method outlined in the journalJ.C.S. Perkin 1, 1981, No. 5, 1520–30. ¹HNMR (300 MHz, CDCl₃) δ 10.51(s, 1H), 8.65 (s,1H), 7.85 (d,J=9 Hz, 1H), 7.37 (s, 1H), 7.27 (d,J=12,1H), 3.99 (s, 3H).

(b) 2-Chloro-7-methoxy-quinoline-3-carboxylic acid

The title compound was prepared using the material from example 1afollowing the method outlined in J. Het. Chem. 1991, 28(5), 1339–40 ESMSm/e [M+H]⁺=238.5.

(c) 2-(3-Chloro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid

The material from example 1b (600 mg, 2.54 mmol) and 3-chloroaniline(275 uL, 2.54 mmol) was heated at 140° C. in 20 mL xylene for 14 h. Thereaction was cooled, evaporated and purified by flash chromatography(silica gel, 20% MeOH in CHCl₃) to give the above titled compound. ESMSm/e [M+H]⁺=329.5.

Example 2 Preparation of7-Chloro-2-(3-chloro-phenylamino)-quinoline-3-carboxylic acid

Following the procedure outlined in Example 1(a)–(c) using3′-chloroacetanilide in step 1(a) and DMSO as the solvent in step (c),the title compound was prepared. ESMS m/e [M+H]⁺=333.5.

Example 3 Preparation of2-(3-Chloro-phenylamino)-7-methylsulfanyl-quinoline-3-carboxylic acid

Following the procedure of Example 1(a)–(c) using 3′-methylthioacetanilide in step 1(a) and DMSO as the solvent in step (c) the titlecompound was prepared. ESMS n/e [M+H]⁺=344.87

Example 4 Preparation of2-(4-Chloro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid

Following the procedure of Example 1(a)–(c) except using DMSO as thesolvent and 4-chloroaniline in step (c), the title compound wasprepared. ESMS m/e [M+H]⁺=329.6.

Example 5 Preparation of2-(3-Chloro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid amide

The material from Example (1) (128 mg, 0.35 mmol) was attached to Rinkamide resin using HBtU. The reaction shook for 48 h and was washed withCH₂Cl₂, MeOH, and DMF. The resin was treated with 95% aq. TFA for 14 hthe resin was filtered off and the liquid was evaporated to give thetitle compound. ESMS m/e [M+H]⁺=328.6.

Example 6 Preparation of2-(4-Chloro-benzylamino)-7-methoxy-quinoline-3-carboxylic acid

Following the procedure of Example 1(a)–(c) except using DMSO as thesolvent and 4-chlorobenzylamine in step (c), the title compound wasprepared. ESMS m/e [M+H]⁺=343.7.

Example 7 Preparation of7-Methoxy-2-(4-phenoxy-phenylamino)-quinoline-3-carboxylic acid

Following the procedure of Example 1(a)–(c), except using DMSO as thesolvent and 4-phenoxyaniline in step (c), the title compound wasprepared. ESMS m/e [M+H]⁺=387.6.

Example 8 Preparation of7-Methoxy-2-(4-morpholin-4-yl-phenylamino)-quinoline-3-carboxylic acid

Following the procedure of Example 1(a)–(c), except using DMSO as thesolvent and 4-morpholinoaniline in step (c), the title compound wasprepared. ESMS m/e [M+H]⁺=380.6.

Example 9 Preparation of 2-(3-Chloro-phenylamino)-quinoline-3-carboxylicacid

Following the procedure of Example 1(a)–(c), using acetanilide in step1(a) and DMSO as the solvent in step (c), the title compound wasprepared. ESMS m/e [M+H]⁺=299.6.

Example 10 Preparation of2-(3-Chloro-phenylamino)-7-methyl-quinoline-3-carboxylic acid

(a) 2-Chloro-3-formyl-7-methyl-quinoline

The title compound was prepared using 3′-methyl acetanilide and themethod outlined in J.C.S. Perkin 1, 1981, No. 5, 1520–30 ¹HNMR (300 MHz,CDCl₃) δ 10.34 (s, 1H), 8.81 (s,1H), 8.14 (d,J=9 Hz, 1H), 7.80 (s, 1H),7.58 (d, J=9,1H), 2.57 (s, 3H).

(b) 2-Chloro-7-methyl-quinoline-3-carboxylic acid

The title compound was prepared using the material from Example 10afollowing the method outlined in J. Het. Chem. 1991, 28(5), 1339–40 LCESMS m/e [M+H]⁺=222.5

c) 2-(3-Chloro-phenylamino)-7-methyl-quinoline-3-carboxylic acid

The material from Example 10b (370 mg 1.67 mol) and 3-chloroanaline (268ul 2.51 mmol) were heated at 140° C. in 5 mL DMSO for 14 hr. Thereaction was cooled, purified by prep. hplc, (YMC CombiPrep ODS-A, 5min. gradient 20–95% CH₃CN/H₂O with 0.1% TFA) ESMS m/e [M+H]⁺=312.95.

Example 11 Preparation of2-(3-Chloro-phenylamino)-6-methoxy-quinoline-3-carboxylic acid

Following the procedure of Example 1(a)–(c), using p-acetanisidide instep 1(a) and DMSO as the solvent in step (c), the title compound wasprepared. ESMS m/e [M+H]⁺=329.6.

Example 12 Preparation of2-(3-Chloro-phenylamino)-7-ethyl-quinoline-3-carboxylic acid

Following the procedure of Example 1(a)–(c), using p-acetanisidide instep 1(a) and DMSO as the solvent in step (c), the title compound wasprepared. LCMS m/e [M+H]⁺=327.2.

Example 13 Preparation of2-(3-Cyano-phenylamino)-7-methoxy-quinoline-3-carboxylic acid

The material from example 1b (250 mg, 1.05 mmol) and 3-aminobenzonitrile(140 mg, 1.1 mmol) in THF (5.5 mL) was treated at −78° C. with 5.5 mL of1.0 M LiN(TMS)₂ in hexane and the resulting solution allowed to warmslowly to room temperature. After 24 h the solvent was evaporated andthe residue purified by preparative hplc to give the above namedcompound. LCMS m/e [M+H]⁺=320.

Example 14 Preparation of7-Methoxy-2-m-tolylamino-quinoline-3-carboxylic acid

Following the procedure of Example 13, with m-toluidine in place of3-aminobenzonitrile gave the above named compound. LCMS m/e [M+H]⁺=309.

Example 15 Preparation of2-(4-Ethoxy-phenylamino)-7-methoxy-quinoline-3-carboxylic acid

Following the procedure of Example 13, with 4-ethoxyaniline in place of3-aminobenzonitrile gave the above named compound. LCMS m/e [M+H]⁺=339.

Example 16 Preparation of2-(4-Cyclohexyl-phenylamino)-7-methoxy-quinoline-3-carboxylic acid

Following the procedure of Example 13, with 4-cyclohexylaniline in placeof 3-aminobenzonitrile gave the above named compound. LCMS m/e[M+H]⁺=377.

Example 17 Preparation of2-(4-Fluoro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid

Following the procedure of Example 13, with 4-fluoroaniline in place of3-aminobenzonitrile gave the above named compound. LCMS m/e [M+H]⁺=313.

Example 18 Preparation of2-(2-Chloro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid

Following the procedure of Example 13, with 2-chloroaniline in place of3-aminobenzonitrile gave the above named compound. LCMS m/e [M+H]⁺=329.

Example 19 Preparation of2-(4-Ethyl-phenylamino)-7-methoxy-quinoline-3-carboxylic acid

Following the procedure of Example 13, with 4-ethylaniline-in place of3-aminobenzonitrile gave the above named compound. LCMS m/e [M+H]⁺=323.

Example 20 Preparation of2-(3-Ethyl-phenylamino)-7-methoxy-quinoline-3-carboxylic acid

Following the procedure of Example 13, with 3-ethylaniline-in place of3-aminobenzonitrile gave the above named compound. LCMS m/e [M+H]⁺=323.

Example 21 Preparation of2-(4-Cyano-phenylamino)-7-methoxy-quinoline-3-carboxylic acid

Following the procedure of Example 13, with 4-aminobenzonitrile-in placeof 3-aminobenzonitrile gave the above named compound. LCMS n/e[M+H]⁺=320.

Example 22 Preparation of7-Methoxy-2-(quinolin-3-ylamino)-quinoline-3-carboxylic acid

Following the procedure of Example 13, with 3-aminoquinoline-in place of3-aminobenzonitrile gave the above named compound. LCMS m/e [M+H]⁺=346.

Example 23 Preparation of2-(4-Iodo-phenylamino)-7-methoxy-quinoline-3-carboxylic acid

Following the procedure of Example 13, with 4iodoaniline-in place of3-aminobenzonitrile gave the above named compound. LCMS m/e [M+H]⁺=421.

Example 24 Preparation of7-Methoxy-2-(pyridin-3-ylamino)-quinoline-3-carboxylic acid

Following the procedure of Example 13, with 3-aminopyridine-in place of3-aminobenzonitrile gave the above named compound. LCMS m/e [M+H]⁺=296.

Example 25 Preparation of7-Methoxy-2-(6-methoxy-pyridin-3-ylamino)-quinoline-3-carboxylic acid

Following the procedure of Example 13, with 5-amino-2-methoxypyridine-inplace of 3-aminobenzonitrile gave the above named compound. LCMS m/e[M+H]⁺=326.

Example 26 Preparation of7-Methoxy-2-(3-acetamino-aminophenyl)-quinoline-3-carboxylic acid

Following the procedure of Example 13, with 3-acetaminoaniline-in placeof 3-aminobenzonitrile gave the above named compound. LCMS m/e[M+H]⁺=352.

Example 27 Preparation of7-Methoxy-2-(quinolin-8-ylamino)-quinoline-3-carboxylic acid

Following the procedure of Example 13, with 8-aminoquinoline-in place of3-aminobenzonitrile gave the above named compound. LCMS m/e [M+H]⁺=346.

Example 28 Preparation of7-Methoxy-2-(quinolin-5-ylamino)-quinoline-3-carboxylic acid

Following the procedure of Example 13, with 5-aminoquinoline-in place of3-aminobenzonitrile gave the above named compound. LCMS m/e [M+H]⁺=346.

Example 29 Preparation of2-(3,4-Dimethoxy-phenylamino)-7-methoxy-quinoline-3-carboxylic acid

Following the procedure of Example 13, with 3,4-dimethoxyaniline-inplace of 3-aminobenzonitrile gave the above named compound. LCMS m/e[M+H]⁺=355.

Example 30 Preparation of2-(3,4-Dimethyl-phenylamino)-7-methoxy-quinoline-3-carboxylic acid

Following the procedure of Example 13, with 3,4-dimethylaniline-in placeof 3-aminobenzonitrile gave the above named compound. LCMS m/e[M+H]⁺=323.

Example 31 Preparation of2-(2-Fluoro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid

Following the procedure of Example 13, with 2-fluoroaniline-in place of3-aminobenzonitrile gave the above named compound. LCMS m/e [M+H]⁺=313.

Example 32 Preparation of2-(4-Ethoxy-2-fluoro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid

Following the procedure of Example 13, with 2-fluoro-3-ethoxyaniline-inplace of 3-aminobenzonitrile gave the above named compound. LCMS m/e[M+H]⁺=357.

Example 33 Preparation of7-Methoxy-2-piperidin-1-yl-quinoline-3-carboxylic acid

Following the procedure of Example 1(a)–(c) except using DMSO as thesolvent and piperidine in step (c), the title compound was prepared.LCMS m/e [M+H]⁺=287.2.

Example 34 Preparation of 2-Propylamino7-methoxy-quinoline-3-carboxylicacid

Following the procedure of Example 1(a)–(c) except using DMSO as thesolvent and propylamine in step (c), the title compound was prepared.LCMS m/e [M+H]⁺=261.0.

Example 35 Preparation of 2-(3-Chloroanilino)--7-ethoxy-quinoline-3-carboxylic acid

(a) 2-Chloro-3-formyl-7-ethoxy-quinoline

Using the method described in Example 1(a), the title compounds wasprepared. LCMS m/e [M+H]⁺=236.

(b) 2-Chloro-7-methoxy-quinoline-3-carboxylic acid

Using the method described in Example 1(b), the title compounds wasprepared. ESMS m/e [M+H]⁺=253.

(c) 2-(3-Chloroanilino)-7-ethoxy-quinoline-3-carboxylic acid

Following the procedure of Example 13, with2-chloro-7-methoxy-quinoline-3-carboxylic acid from 35b and3-chloroaniline-in place of 3-aminobenzonitrile gave the above namedcompound LCMS m/e [M+H]⁺=343.

Example 36 Preparation of 2-Methyl 7-methoxy-quinoline-3-carboxylic acid

(a)2-Methyl-7-methoxy-2quinoline-3-carboxylic acid ethyl ester The titlecompound was prepared using the method outlined in SyntheticCommunications 1987, 17 (14), 1647–1653. LCMS m/e [M+H]⁺=246.2.

(b) 2-Methyl 7-methoxy-quinoline-3-carboxylic acid The material fromabove (2 g) was dissolved in ethanol and was treated with 9 mL 1N NaOH(aq). The reaction stirred at rt for 12 h. The reaction was evaporatedand suspended in CHCl₃. The product was precipitated out with 1N HCl(aq). LCMS m/e [M+H]⁺=218.2.

Example 37 Preparation of 2,7-Dimethyl-quinoline-3-carboxylic acid

Following the procedure of Example 36 (a)-(b) substituting m-toluidinein step 36 (a) gave the above named compound. LCMS m/e [M+H]⁺=202.0.

Example 38 Preparation of 7-Methoxy-2-phenyl-quinoline-3-carboxylic acid

Following the procedure of Example 36 (a)-(b) substitutingethylbenzoylacetate in step 36 (a) gave the above named compound. LCMSm/e [M+H]⁺=280.2

The above specification and Examples fully disclose how to make and usethe compounds of the present invention. However, the present inventionis not limited to the particular embodiments described hereinabove, butincludes all modifications thereof within the scope of the followingclaims. The various references to journals, patents and otherpublications which are cited herein comprise the state of the art andare incorporated herein by reference as though fully set forth.

1. A compound of Formula I:

wherein: R₁ is selected from the group consisting of: —NH—C₁₋₆alkyl,—NH—C₃₋₇ cycloalkyl, —NH—C₃₋₇ cycloheteroalkyl, —NH-aryl, —NH-Het,—O—C₁₋₆alkyl, —O—C₃₋₇ cycloalkyl, —O—C₃₋₇ cycloheteroalkyl, —O-aryl,—O-Het, —S—C₁₋₆ alkyl, —S—C₃₋₇ cycloalkyl, —S—C₃₋₇ cycloheteroalkyl,—S-aryl, —S-Het, —C₃₋₇ cycloalkyl and —C₃₋₇ cycloheteroalkyl; R₂ isselected from the group consisting of: —CO₂H, —CONH₂, and —CO₂R′; R₃ isselected from the group consisting of: —H, —OH, —C₁₋₆alkyl, —C₃₋₇cycloalkyl, aryl, Het, —O—C₁₋₆alkyl, —O—C₃₋₇ cycloalkyl, —O-aryl,—O-Het, —S—C₁₋₆alkyl, —S—C₃₋₇ cycloalkyl, —S-aryl, —S-Het,—NH—C₁₋₆alkyl, —NH—C₃₋₇ cycloalkyl, —NH-aryl, —NH-Het and halogen; R₄ isselected from the group consisting of: —H, —C₁₋₆alkyl, —C₃₋₇ cycloalkyl,aryl, Het, —O—C₁₋₆alkyl, —O—C₃₋₇ cycloalkyl, —O-aryl, —O-Het,—S—C₁₋₆alkyl, —S—C₃₋₇ cycloalkyl, —S,-aryl, —S-Het, —NH—C₁₋₆alkyl,—NH—C₃₋₇ cycloalkyl, —NH-aryl, —NH-Het and halogen; R₃ and R₄ can form a5 to 7 membered ring comprising 0–3 heteroatoms independently selectedfrom the group consisting of: O, N, and S; R₅ is selected from the group—H and halogen; R′ is selected from the group consisting of: —C₁₋₆alkyl,—C₃₋₇cycloalkyl, and —C₃₋₇cycloheteroalkyl; and R″ is selected from thegroup consisting of: —C₁₋₆alkyl, —C₃₋₇cycloalkyl, —C₃₋₇cycloheteroalkyl,aryl, and Het; or a pharmaceutically acceptable salt, hydrate or solvatethereof.
 2. A compound according to claim 1 wherein: R₁ is selected fromthe group consisting of: —NH—C₁₋₆alkyl, —NH-aryl, —NH-Het, —O-aryl,—O-Het, —S-aryl, —S-Het, and —C₃₋₇ cycloheteroalkyl, R₃ is selected fromthe group consisting of: —H, —C₁₋₆alkyl, —O—C₁₋₆alkyl, —S—C₁₋₆alkyl, andhalogen; and R₄ is —O—C₁₋₆alkyl, —S—C₁₋₆alkyl, and halogen.
 3. Acompound according to claim 2 wherein, when R₂ is —CO₂R, R′ is selectedfrom the group consisting of: —C₁₋₆alkyl and —C₃₋₇cycloalkyl.
 4. Acompound according to claim 2 wherein: R₁ is selected from the groupconsisting of: —NH—C₁₋₆alkyl, —NH-aryl, —NH-Het, and —C₃₋₇cycloheteroalkyl; R₂ is selected from the group consisting of: —CO₂H and—CONH₂; R₃ is selected from the group consisting of: —H, —C₁₋₆alkyl,—O—C₁₋₆alkyl, —S—C₁₋₆alkyl, and halogen; and R₄ is selected from thegroup consisting of: —H and halogen; and R₅ is —H.
 5. A compoundaccording to claim 4 wherein R₄ is H.
 6. A compound according to claim 4wherein: —NH-aryl is selected from the group consisting of:3-methylphenylamino, 3-ethylphenylamino, 4-ethylphenylamino,4-cyclohexylphenylamino, 3,4-dimethylphenylamino, 2-chlorophenylamino,3-chlorophenylamino, 4-chlorophenylamino, 2-fluorophenylamino,4-fluorophenylamino;, 4-iodophenylamino, 4-chlorobenzylamino,4-morpholin-4-yl-phenylamino, 3-cyanophenylamino, 4-cyanophenylamino,4-ethoxyphenylamino, 3,4-dimethoxyphenylamino, 4-phenoxyphenylamino and2-fluoro-3-ethoxyphenylamino; —NH-Het selected from the group consistingof: quinolin-3-ylamino, quinolin-5-ylamino, quinolin-8-ylamino,pyridin-3-ylamino and 6-methoxy-pyridin-3-ylamino; —C₃₋₇cycloheteroalkyl is N-piperidino; and —NH—C₁₋₆alkyl is 2-propylamino;and in R₃: —C₁₋₆alkyl is selected from the group consisting of :methyland ethyl; —O—C₁₋₆alkyl is methoxy; —S—C₁₋₆alkyl is methylsulfanyl; andhalogen is chloro.
 7. A compound according to claim 6 wherein thecompound of Formula I is selected from the group consisting of:2-(3-chloroanilino)-3-carboxy-7-methoxy-quinoline;2-(3-chloroanilino)-3-carboxy-7-chloro-quinoline;2-(3-chloroanilino)-3-carboxy-7-methylthio-quinoline;2-(4-chloroanilino)-3-carboxy-7-methoxy-quinoline;2-(3-chloroanilino)-3-carboxamido-7-methoxy-quinoline;2-(4-chlorobenzylamino)-3-carboxy-7-methoxy-quinoline;2-(4-phenoxyanilino)-3-carboxy-7-methoxy-quinoline;2-(4-morpholinanilino)-3-carboxy-7-methoxy-quinoline;2-(3-chloroanilino)-3-carboxy-quinoline;2-(3-chloroanilino)-3-carboxy-7-methyl-quinoline;2-(3-chloroanilino)-3-carboxy-6-methoxy-quinoline;2-(3-chloroanilino)-3-carboxy-7-ethyl-quinoline;2-(3-cyanoanilino)-3-carboxy-7-methoxy-quinoline;2-(3-methylanilino)-3-carboxy-7-methoxy-quinoline;2-(4-ethoxyanilino)-3-carboxy-7-methoxy-quinoline;2-(4-cyclohexylanilino)-3-carboxy-7-methoxy-quinoline;2-(4-fluoroanilino)-3-carboxy-7-methoxy-quinoline;2-(2-chloroanilino)-3-carboxy-7-methoxy-quinoline;2-(4-ethylanilino)-3-carboxy-7-methoxy-quinoline;2-(3-ethylanilino)-3-carboxy-7-methoxy-quinoline;2-(4-cyanoanilino)-3-carboxy-7-methoxy-quinoline;2-(3-aminoquinolino)-3-carboxy-7-methoxy-quinoline;2-(4-iodoanilino)-3-carboxy-7-methoxy-quinoline;2-(3-aminopyridino)-3-carboxy-7-methoxy-quinoline;2-(5-amino-2-methoxypyridino)-3-carboxy-7-methoxy-quinoline;2-(8-aminoquinolino)-3-carboxy-7-methoxy-quinoline;2-(5-aminoquinolino)-3-carboxy-7-methoxy-quinoline;2-(3,4-dimethoxyanilino)-3-carboxy-7-methoxy-quinoline;2-(3,4-dimethylanilino)-3-carboxy-7-methoxy-quinoline;2-(2-fluoroanilino)-3-carboxy-7-methoxy-quinoline;2-(2-fluoro-3-ethoxyanilino)-3-carboxy-7-methoxy-quinoline;2-piperidino-3-carboxy-7-methoxy-quinoline; and2-propylamino-3-carboxy-7-methoxy-quinoline.
 8. A compound according toclaim 7 wherein the compound of Formula I is selected from the groupconsisting of: 2-(3-Chloro-phenylamino)-7-methoxy-quinoline-3-carboxylicacid; 7-Chloro-2-(3-chloro-phenylamino)-quinoline-3-carboxylic acid;2-(3-Chloro-phenylamino)-7-methylsulfanyl-quinoline-3-carboxylic acid;2-(4-Chloro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;2-(4-Chloro-benzylamino)-7-methoxy-quinoline-3-carboxylic acid;7-Methoxy-2-(4-phenoxy-phenylamino)-quinoline-3-carboxylic acid;2-(3-Cyano-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;7-Methoxy-2-m-tolylamino-quinoline-3-carboxylic acid;2-(4-Ethoxy-phenylamino)-7-methoxy-quinoline-3-carboxylic acid2-(4-Cyclohexyl-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;2-(4-Fluoro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;2-(2-Chloro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;2-(4-Ethyl-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;2-(3-Ethyl-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;2-(4-Cyano-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;7-Methoxy-2-(quinolin-3-ylamino)-quinoline-3-carboxylic acid;2-(4-lodo-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;7-Methoxy-2-(6-methoxy-pyridin-3-ylamino)-quinoline-3-carboxylic acid;7-Methoxy-2-(quinolin-8-ylamino)-quinoline-3-carboxylic acid;7-Methoxy-2-(quinolin-5-ylamino)-quinoline-3-carboxylic acid;2-(3,4-Dimethoxy-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;2-(2-Fluoro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid; and2-(4-Ethoxy-2-fluoro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid.9. A compound according to claim 8 wherein the compound of Formula I isselected from the group consisting of:2-(3-Chloro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;2-(3-Chloro-phenylamino)-7-methylsulfanyl-quinoline-3-carboxylic acid;2-(4-Chloro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;2-(3-Cyano-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;7-Methoxy-2-m-tolylamino-quinoline-3-carboxylic acid;2-(4-Ethoxy-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;2-(4-Fluoro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;2-(2-Chloro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;2-(4-Ethyl-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;2-(3-Ethyl-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;2-(4-Cyano-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;2-(4-lodo-phenylamino)-7-methoxy-quinoline-3-carboxylic acid;7-Methoxy-2-(quinolin-8-ylamino)-quinoline-3-carboxylic acid;7-Methoxy-2-(quinolin-5-ylamino)-quinoline-3-carboxylic acid;2-(2-Fluoro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid; and2-(4-Ethoxy-2-fluoro-phenylamino)-7-methoxy-quinoline-3-carboxylic acid.